Vascular embolic system

ABSTRACT

Systems and methods of blocking a biological vessel are provided. The systems and methods may comprise introducing to the vessel an amphiphilic peptide. The peptide may comprise at least thirteen amino acids that may alternate between a hydrophobic amino acid and a hydrophilic amino acid. The peptide may form a beta-sheet spontaneously in an aqueous solution in the presence of a cation.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 14, 2013, isnamed T2071-7000WO_SL.txt and is 23,527 bytes in size.

FIELD OF THE DISCLOSURE

This disclosure relates to macroscopic membranes that may be used inmedical, research, and industrial applications. More particularly, thisdisclosure relates to membranes, hydrogels, compositions and solutionsthat may be used in a vascular embolic system and embolizationprocedures. The vascular embolic system may provide an approach to atleast partially block biological pathways or channels including vessels,veins, portal veins, arteries, and ducts that may transport blood andother fluids, such as lymph fluids.

SUMMARY

A method of blocking a biological vessel in a subject is provided. Themethod comprises introducing a catheter into a biological vessel andpositioning an end of the catheter in a target area of the biologicalvessel in which at least a partial obstruction is desired. The methodfurther comprises administering through the catheter a solutioncomprising an amphiphilic peptide comprising at least 12 amino acidsthat alternate between a hydrophobic amino acid and a hydrophilic aminoacid in an effective amount and in an effective concentration to form ahydrogel under physiological conditions to allow at least partialblockage of the biological vessel. The method further comprises removingthe catheter from the biological vessel with the at least partialobstruction in place.

A kit for blocking a biological vessel in a subject is provided. The kitcomprises a solution comprising an amphiphilic peptide comprising atleast 12 amino acids that alternate between a hydrophobic amino acid anda hydrophilic amino acid in an effective amount and in an effectiveconcentration to form a hydrogel under physiological conditions to allowat least partial blockage of the biological vessel. The kit furthercomprises instructions for administering the solution to the biologicalvessel in the subject.

A method of facilitating blocking a biological vessel in a subject isprovided. The method comprises providing a solution comprising anamphiphilic peptide comprising at least 12 amino acids that alternatebetween a hydrophobic amino acid and a hydrophilic amino acid in aneffective amount and in an effective concentration to form a hydrogelunder physiological conditions to allow at least partial blockage of thebiological vessel. The method further comprises providing instructionsfor administering the solution to a target area of the biological vesselthrough introduction of the solution to a catheter positioned in thebiological vessel.

BRIEF DESCRIPTION OF THE DRAWING

The accompanying drawings are not intended to be drawn to scale. Forpurposes of clarity, not every component may be labeled.

In the drawings:

FIG. 1 is an image of a cross-section of a portal vein embolism using apeptide solution of the present disclosure;

FIG. 2 is a contrast image of a normal hepatic artery;

FIG. 3 is a contrast image of an injection of the materials of thepresent disclosure;

FIG. 4 is a contrast image of a hepatic artery after injection with thematerials of the present disclosure;

FIG. 5 is a contrast image of a hepatic artery two weeks after injectionwith the materials of the present disclosure;

FIG. 6A is a histopathological image of a peptide hydrogel located in ahepatic artery;

FIG. 6B is a histopathological image of peptide hydrogel located in ahepatic artery;

FIG. 6C is a histopathological image of peptide hydrogel located in ahepatic artery; and

FIG. 7 is a hepatic cell necrosis image of a peptide hydrogel embolismlocation;

FIG. 8 is an image of an artery before embolization; and

FIG. 9 is an image of an artery after embolization.

DETAILED DESCRIPTION

Embolization is a procedure that creates a blockage, lodging, occlusion,or embolism in one or more biological pathways or channels. Thebiological pathways or channels may include vessels, veins, portalveins, arteries, and ducts that may transport blood and other fluids,such as lymph fluids. Embolization is used to treat a wide variety ofconditions affecting different organs of a subject's body, including thehuman body. The one or more vessels may be targeted to purposely preventor reduce the circulation of blood to a desired target. The embolizationprocedure may be used to purposely create such a blockage, lodging orocclusion in order to deprive tumors or other pathological processes oftheir blood supply (perfusion). Embolization may be used to treatdisorders, malformations, or congenital ailments in biological vessels.For example, embolization may be used to treat patent ductus arteriosus(PDA). The embolization treatment may be used to treat majoraortopulmonary collateral artery (MAPCA), recurrent hemotysis,arteriovenous malformations, cerebral aneurysms, gastrointestinalbleeding, epistaxis, post-partum hemorrhage, surgical hemorrhage, anduterine fibroids.

Embolization may be accomplished by several different techniques. It maybe accomplished by administering a material, such as a liquid to adesired or predetermined location, such as a target area. Administeringmay include applying or injecting a material, such as a liquid to adesired or predetermined location, such as a target area.

Embolization may be used to shut down or block all or a portion of avessel which forms an aneurysm in order to prevent the aneurysm fromrupturing. Embolization may also be used to shut down or block all or aportion of certain blood vessels that surround a region of a subjectthat is being operated on, for example, during surgery of cerebralarteriovenous malformation (AVM).

Obstructing materials may be used to create a blockage or occlusion in abiological vessel to accomplish embolization. Obstructing materials mayinclude metal coils, collagens, cyanoacrylates, and other materials. Thematerials may be inserted or placed at the desired surgical sites withthe use of a catheter. Balloons may also be implanted in a target vesseland filled with saline.

Metal coils may remain in vivo permanently, but the safety of thesecoils in long-term applications is unknown. The metal coils may alsoresult in incompatibility with magnetic devices. Collagens may havebiological incompatabilities and cyanoacrylates may become toxic invivo.

Specific liquid embolization agents may include onyx,n-butyl-2-cyanoacrylate (nbca) and ethiodol, made from iodine, poppyseedoil. Schlerosing agents, which harden the endothelial lining of vesselsmay also be used. Examples of such agents include ethanol, ethanolamineoleate and sotradecol. Particulate embolization agents include polyvinylalcohol and acrylic gelatin microspheres.

The present disclosure provides for methods of embolizing or blockingbiological vessels, methods of facilitating blocking a biologicalvessel, and kits for use in blocking a biological vessel. Biologicalvessels may include blood vessels and lymph ducts. Blood vessels mayinclude arteries, veins, portal veins and capillaries. The term vascularmay refer to biological vessels, including arteries, veins, portalveins, capillaries, and ducts.

The methods may comprise blocking or obstructing a biological vessel ina subject or methods of facilitating blocking or obstructing abiological vessel in a subject. As used herein, the term “subject” isintended to include human and non-human animals, for example,vertebrates, large animals, and primates. In certain embodiments, thesubject is a mammalian subject, and in particular embodiments, thesubject is a human subject. Although applications with humans areclearly foreseen, veterinary applications, for example, with non-humananimals, are also envisaged herein. The term “non-human animals” of theinvention includes all vertebrates, for example, non-mammals (such asbirds, for example, chickens; amphibians; reptiles) and mammals, such asnon-human primates, domesticated, and agriculturally useful animals, forexample, sheep, dog, cat, cow, pig, rat, among others.

The embolism, blockage or obstruction may be partial or complete. Bycomplete it is meant that the embolism, blockage or obstruction preventssubstantially all blood flow past the embolism, blockage, orobstruction. The systems and methods may include administration,application, or injection of a self-assembling peptide, or a solutioncomprising a self-assembling peptide, to a predetermined or desiredtarget area. The self-assembling peptide may be applied or introduced toa biological vessel in the form of a self-assembling peptide solution,hydrogel, membrane or other form.

The self-assembling peptide solution may be an aqueous self-assemblingpeptide solution. The self-assembling peptide, also referred to hereinas “peptide” or “amphiphilic peptide” may be administered, applied, orinjected in a solution that is substantially cell-free.

In certain embodiments, the self-assembling peptide may be administered,applied, or injected in a solution that is cell-free.

The self-assembling peptide may also be administered, applied orinjected in a solution that is substantially drug-free. In certainembodiments, the self-assembling peptide may be administered, applied,or injected in a solution that is drug-free. In certain otherembodiments, the self-assembling peptide may be administered, applied,or injected in a solution that is substantially cell-free andsubstantially drug-free. In still further certain other embodiments, theself-assembling peptide may be administered, applied, or injected in asolution that is cell-free and drug-free.

Administration of a solution may comprise, consist of, or consistessentially of administration of a solution comprising, consisting of,or consisting essentially of an amphiphilic peptide comprising,consisting of, or consisting essentially of at least 12 amino acids thatalternate between a hydrophobic amino acid and a hydrophilic amino acid.

The systems and methods may comprise administering a self-assemblingpeptide to a predetermined or desired target as a hydrogel. A hydrogelis a term that may refer to a colloidal gel that is dispersed in water.The systems and methods may also comprise applying a self-assemblingpeptide to a predetermined or desired target as a solution, such as anaqueous peptide solution.

When using the term “administering,” it is intended to include, but isnot limited to, applying, introducing or injecting the self-assemblingpeptide, in one or more of various forms including, but not limited to,by itself, by way of a solution, such as an aqueous solution, or by wayof a hydrogel, with or without additional components.

The method of blocking the biological vessel in a subject may compriseintroducing a syringe, pipette, catheter, or other needle-based deviceinto the biological vessel. The self-assembling peptide may beadministered by way of a syringe, pipette, catheter, or otherneedle-based device into the biological vessel. The gauge of the syringeneedle may be selected to provide an adequate flow of liquid from thesyringe to the target area. This may be based in some embodiments on atleast one of the amount of self-assembling peptide or peptide solutionbeing administered, the concentration of the peptide in solution, andthe viscosity of the peptide solution.

The method of blocking the biological vessel in the subject may compriseintroducing a catheter into the biological vessel and positioning an endof the catheter in a target area of the biological vessel in which atleast a partial obstruction is desired. The self-assembling peptide maybe administered by way of a catheter to the target area of a biologicalvessel in which at least a partial obstruction is desired. The use of acatheter may provide a more selective administration of the peptide toprovide for a more accurate delivery to the target area. Selectiveadministration of the peptide may allow for enhanced and more targeteddelivery of the peptide solution such that blockage of the biologicalvessel is successful and positioned in the desired location in anaccurate manner. The selective administration may provide enhanced,targeted delivery that markedly improves the positioning andeffectiveness of the blockage in the biological vessel over use of asyringe or other means.

Use of the catheter may include use of accompanying devices, such as aguidewire used to guide the catheter into position. The guidewire may beintroduced into the biological vessel prior to introducing the catheter.Once the administration of the peptide solution is complete, or once theat least partial obstruction or blockage is in place, the catheter maybe removed from the biological vessel.

The use of a syringe, needle, pipette, other needle-based device, orcatheter may require determining the diameter of the biological vesselwhich is targeted, such that at least a portion of the syringe, needle,pipette, other needle-type device, or catheter may enter the biologicalvessel to administer the peptide, peptide solution, or hydrogel to thetarget area.

In certain embodiments, the hydrogel may be formed in vitro andadministered to the desired location in vivo. In certain examples, thislocation may be the area in which it is desired to create an embolism.In other examples, this location maybe upstream or downstream of thearea in which it is desired to form an embolism. In this case, it may bedesired to allow an unassisted movement or migration of the hydrogel tothe area in which it is desired to form an embolism. Alternatively,another procedure may position the hydrogel in the area in which it isdesired to form an embolism. The desired location or target area may bea portion of a biological vessel. The desired location or target areamay be a portion within a biological vessel.

In certain aspects of the disclosure, the hydrogel may be formed invivo. A solution comprising the self-assembling peptide, such as anaqueous solution, may be inserted to an in vivo location or area of asubject to allow an embolism to be created at that location. In certainexamples, the hydrogel may be formed in vivo at one location, andallowed to move the hydrogel unassisted to the area in which it isdesired to form an embolism. Alternatively, another procedure may placethe hydrogel in the area in which it is desired to form an embolism. Thepeptides of the present disclosure may be in the form of a powder, asolution, a gel, or the like. Since the self-assembling peptide gels inresponse to changes in solution pH and salt concentration, it can bedistributed as a liquid that gels upon contact with a subject duringapplication or administration.

The particular self-assembling peptides of the present disclosure mayprovide for improved adhesion to tissue over other agents that may beused in biological vessel embolization. The improved adhesion may be dueto the composition of the peptide (for example, the particular aminoacids of the peptide), the structure of the peptide once self-assembled,or due to the self-assembly process itself. In certain embodiments, itmay benefit the procedure to remove excess body fluid, such as blood orbile, from the target site or area in which it is desired to provide ahydrogel for occlusion.

In some embodiments, the peptide or hydrogel may not adhere to thetissue or biological vessel. As the peptide or peptide solution isadministered, it comes in contact with the blood or other fluid in thebiological vessel, which causes gelation inside the vessel. The peptidesolution can move within the vessel, but as it begins to gel it losesits fluidity and will remain in position at a position or target area inthe biological vessel. The peptides in the form of a hydrogel may remainin place without adhesion to the tissue or biological vessel.

This disclosure relates to aqueous solutions, hydrogels, and membranescomprising self-assembling peptides, sometimes referred to asself-assembling oligopeptides, or amphiphilic peptides. The peptides maybe comprised of an amphiphilic peptide having about 6 to about 200 aminoacid residues with the hydrophilic amino acids and hydrophobic aminoacids alternately bonded. The self-assembling peptides may exhibit abeta-structure in aqueous solution in the presence of physiological pHand/or a cation, such as a monovalent cation.

The order of effectiveness of the monovalent cations appears to beLi⁺>Na⁺>K⁺>Cs⁺. Cs⁺ may produce the least amount of membranes and inaddition, yields nonmembranous precipitates. The effectiveness of themonovalent cations may correlate inversely with the crystal radii of theions: Li⁺ (0.6 Angstroms), Na⁺ (0.95 Angstroms), K⁺ (1.33 Angstroms),and Cs⁺ (1.69 Angstroms) (Pauling, 1960). A correlation may also be seenwith the hydrated radii of the ions: Li⁺ (3.4 Angstroms), Na⁺ (2.76Angstroms), K⁺ (2.32 Angstroms), and Cs⁺ (2.28 Angstroms), and with theorder of enthalpies of the monovalent cations (Pauling, 1960). Thepresence of the the monovalent metal cations may act as a catalyst ormay be incorporated into the membrane. The size of the filaments (10-20nm) and interfilament distance (50-80 nm) in some membranes formed maysuggest that hydrated ions may stabilize the intermolecular interaction.Some anions, including divalent anions, acetate, Cl⁻, SO₄ ⁻², and PO₄⁻², and organic ions, NH₄ ⁺ and Tris-Cl, may not induce membraneformation.

Concentrations of monovalent metal cations (NaCl) as low as 5 mM and ashigh as 5M have been found to induce membrane formation within a fewminutes in certain embodiments. Thus, membrane formation may beindependent of salt concentration over this wide range. Saltconcentrations of less than 5 mM may also induce membrane formation, butat a slower rate.

The peptides may be generally stable in aqueous solutions andself-assemble into large, extremely stable macroscopic structures ormatrices when exposed to physiological conditions or levels of salt. Thepresence of a monovalent alkali metal ion such as sodium ions andpotassium ions present at physiological levels promote formation of ahydrogel from the peptide solution. Once the hydrogel is formed it maynot decompose even under common protein denaturing conditions such ashigh temperature or with denaturing agents such as acids, alkalis,proteases, urea, guanidine hydrochloride or the like. The self-assembledpeptides may be visible to the naked eye when stained with a dye, CongoRed, and can form sheet-like or fibril structures which have hightensile strength. These materials are substantially resistant to changein pH, heat, and enzymatic proteolysis. The self-assembled peptides havea fibrous microstructure with small pores as revealed by electronmicroscopy.

Physiological conditions may occur in nature for a particular organismor cell system, which may be in contrast to artificial laboratoryconditions. The conditions may comprise one or more properties such asone or more particular properties or one or more ranges of properties.For example, the physiological conditions may include a temperature orrange of temperatures, a pH or range of pH's, a pressure or range ofpressures, and one or more concentrations of particular compounds,salts, and other components. For example, in some examples, thephysiological conditions may include a temperature in a range of about20 to about 40 degrees Celsius. In some examples, the atmosphericpressure may be about 1 atm. The pH may be in a range of about 6 toabout 8. The physiological conditions may include cations such asmonovalent metal cations that may induce membrane formation. These mayinclude sodium chloride (NaCl). The physiological conditions may alsoinclude a glucose concentration, sucrose concentration, or other sugarconcentration, of between about 1 mM and about 20 mM.

The self-assembling peptides of the present disclosure may have at least8 amino acids, at least 12 amino acids, or at least 16 amino acids. Thepeptides may also be complementary and structurally compatible.Complementary refers to the ability of the peptides to interact throughionized pairs and/or hydrogen bonds which form between their hydrophilicside-chains, and structurally compatible refers to the ability ofcomplementary peptides to maintain a constant distance between theirpeptide backbones. Peptides having these properties participate inintermolecular interactions which result in the formation andstabilization of beta-sheets at the secondary structure level andinterwoven filaments at the tertiary structure level.

Both homogeneous and heterogeneous mixtures of peptides characterized bythe above-mentioned properties may form stable macroscopic membranes,filaments, and hydrogels. Peptides which are self-complementary andself-compatible may form membranes in a homogeneous mixture.Heterogeneous peptides, including those which cannot form membranes inhomogeneous solutions, which are complementary and/or structurallycompatible with each other may also self-assemble into macroscopicmembranes, filaments, and hydrogels.

Macroscopic membranes, filaments, and hydrogels formed of theself-assembling peptides may be stable in aqueous solution, in serum,and in ethanol, and may be highly resistant to degradation by heat,alkaline and acidic pH (stable at pH 1.5-11), chemical denaturants (forexample, guanidine-HCl, urea and sodium dodecyl sulfate), and proteasesin vitro (for example, trypsin, alpha-chymotrypsin, papain, protease K,and pronase). They may be non-cytotoxic.

The methods and methods of facilitating of the present disclosure maycomprise administering or providing instructions for administeringthrough a catheter a solution comprising an amphiphilic peptidecomprising at least 12 amino acids that alternate between a hydrophobicamino acid and a hydrophilic amino acid in an effective amount and in aneffective concentration to form a hydrogel under physiologicalconditions to allow at least partial blockage of a biological vessel.

The methods of facilitating may comprise providing the solutioncomprising an amphiphilic peptide comprising the at least 12 amino acidsthat alternate between a hydrophobic amino acid and a hydrophilic aminoacid in an effective amount and in an effective concentration to form ahydrogel under physiological conditions to allow at least partialblockage of a biological vessel.

The methods and methods of facilitating may comprise adding a contrastagent to the peptide solution or providing instructions to add acontrast agent to the solution. Alternatively, the peptide solution maybe manufactured with a contrast agent. The contrast agent may provide avisual image during use of X-ray techniques, such as fluoroscopy orangiography. A nonionic radiopaque contrast media may be included, suchas, for example, a water-soluble iodine based solution. Thewater-soluble iodine based solution may be iopamidol.

The methods and methods of facilitating of the present disclosure maycomprise visualizing a region comprising at least a portion of thebiological vessel or providing instructions to visualize a regioncomprising at least a portion of the biological vessel. Thevisualization may occur during at least one of identifying the targetarea, introducing the catheter, positioning the end of the catheter inthe target area, administering of the solution, and observing thebiological vessel after removing the catheter.

The visualizing may be accomplished through imaging using X-rayradiography. Methods and methods of facilitating may comprisevisualizing or providing instructions to visualize the region usingX-ray radiography. Visualizing may occur for a period of time afteradministering the peptide or removing the catheter. For example, it mayoccur for up to 5 minutes or an hour after administering the peptide orremoving the catheter. Visualization may also occur after one or morepre-determined intervals. For example, visualization may occur about 24hours after administering the peptide or removing the catheter, afterabout one week, after about two weeks, or after about four weeks.Visualization may occur after about 3 months. Visualization may alsooccur after about 6 months. Instructions may be provided to visualizethe region at any one or more of the times disclosed herein and for anyperiod of time. For example, at one week, the visualization may occurfor 1 minute or 5 minutes. At four weeks, the visualization may occurfor 10 minutes or 3 minutes.

Visualizing or monitoring the area surrounding the formed blockage mayalso occur for a period of time or at one or more pre-determinedintervals after administering the peptide or removing the catheter. Thismay occur to determine any one or more of the effectiveness of theblockage, any degradation of the blockage, and any cell or tissuenecrosis.

The methods of the present disclosure may further comprise evaluatingthe subject to determine a need for blocking a biological vessel andpreparing the peptide solution. Preparing the peptide solution maycomprise adding a contrast agent to a preliminary solution comprisingpeptides.

The method of facilitating may comprise providing instructions to add acontrast agent to the solution. The method of facilitating maycomprising providing instructions to combine a sufficient quantity orvolume of the contrast agent in order to adequately do at least one of:identify the target area, introduce a catheter or other administrationdevice, position an end of the catheter in the target area, administerthe peptide solution, remove the catheter or other administrationdevice, and observe the biological vessel after removing the catheter.The use of the contrast agent may allow visualization of the area towhich the peptide, peptide solution or hydrogel is administered.

The amino acids of the self-assembling or amphiphilic peptides may beselected from d-amino acids, l-amino acids, or combinations thereof. Thehydrophobic amino acids include Ala, Val, Ile, Met, Phe, Tyr, Trp, Ser,Thr and Gly. The hydrophilic amino acids can be basic amino acids, forexample, Lys, Arg, His, Orn; acidic amino acids, for example, Glu, Asp;or amino acids which form hydrogen bonds, for example, Asn, Gln. Acidicand basic amino acids may be clustered on a peptide. The carboxyl andamino groups of the terminal residues may be protected or not protected.Membranes may be formed in a homogeneous mixture of self-complementaryand self-compatible peptides or in a heterogeneous mixture of peptideswhich are complementary and structurally compatible to each other.Peptides fitting the above criteria may self-assemble into macroscopicmembranes under suitable conditions, described herein.

The peptides of the present disclosure may include peptides having therepeating sequence of arginine, alanine, aspartic acid and alanine(Arg-Ala-Asp-Ala (RADA) (SEQ ID NO: 1)), and such peptide sequences maybe represented by (RADA)_(p), wherein p=2-50 (SEQ ID NO: 2). Otherpeptide sequences may be represented by self-assembling peptides havingthe repeating sequence of isoleucine, glutamic acid, isoleucine andlysine (Ile-Glu-Ile-Lys (IEIK) (SEQ ID NO: 3)), and such peptidesequences are represented by (IEIK)_(p), wherein p=2-50 (SEQ ID NO: 4).Other peptide sequences may be represented by self-assembling peptideshaving the repeating sequence of lysine, leucine, aspartic acid, andleucine (Lys-Leu-Asp-Leu (KLDL) (SEQ ID NO: 5)), and such peptidesequences are represented by (KLDL)_(p), wherein p=2-50 (SEQ ID NO: 6).The self-assembling peptides may be composed of about 8 to about 200amino acid residues. In certain embodiments, about 8 to about 32residues may be used in the self-assembling peptides, while in otherembodiments self-assembling peptides may have about 12 to about 17residues. The peptides may have a length of about 5 nm.

As specific examples of self-assembling peptides according to theinvention there may be a self-assembling peptide RADA16 having thesequence Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala(RADA)₄ (SEQ ID NO: 7), a self-assembling peptide IEIK13 having thesequence Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile (IEIK)₃I(SEQ ID NO: 8), a self-assembling peptide IEIK17 having the sequenceIle-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile(IEIK)₄I (SEQ ID NO: 9) or a self-assembling peptide KLDL12 having thesequence Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu (KLDL)₃ (SEQ IDNO: 10). A 1% aqueous solution of (RADA)₄ (SEQ ID NO: 7) is available asthe product PuraMatrix™ by 3D-Matrix Co., Ltd. PuraMatrix™ contains 1%peptide having the sequence (RADA)₄ (SEQ ID NO: 7), in water.

Certain peptides may contain sequences which are similar to the cellattachment ligand RGD (Arginine-Glycine-Aspartic acid). The suitabilityof these peptides for supporting in vitro cell growth was tested byintroducing a variety of cultured primary and transformed cells tohomopolymer sheets ofAla-Glu-Ala-Glu-Ala-Lys-Ala-Lys-Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys(AEAEAKAKAEAEAKAK (EAK16) (SEQ ID NO: 11), RAD 16 (SEQ ID NO: 22),RADA16 (SEQ ID NO: 7), and heteropolymers of RAD16 (SEQ ID NO: 22) andEAK16 (SEQ ID NO: 11). The RAD-based peptides may be of particularinterest because the similarity of this sequence to RGD. The RADsequence is a high affinity ligand present in the extracellular matrixprotein tenascin and is recognized by integrin receptors. The EAK 16peptide (SEQ ID NO: 11) and other peptides disclosed herein were derivedfrom a region of a yeast protein, zuotin.

The self-assembly of the peptides may be attributable to hydrogenbonding and hydrophobic bonding between the peptide molecules by theamino acids composing the peptides.

The self-assembling peptides of the present disclosure may have ananofiber diameter in a range of about 10 nm to about 20 nm and anaverage pore size is in a range of about 5 nm to about 200 nm. Incertain embodiments, the nanofiber diameter, the pore size, and thenanofiber density may be controlled by at least one of the concentrationof peptide solution used and the amount of peptide solution used, suchas the volume of peptide solution. As such, at least one of a specificconcentration of peptide in solution and a specific amount of peptidesolution to provide at least one of a desired nanofiber diameter, poresize, and density to adequately deliver and form an embolism uponadministration to a biological vessel may be selected.

As used herein, an amount of a peptide, peptide solution or hydrogeleffective to provide at least a partial obstruction, blockage, orocclusion or treat a disorder, an “effective amount” or a“therapeutically effective amount” refers to an amount of the peptide,peptide solution or hydrogel, which is effective, upon single ormultiple administration (application or injection) to a subject, intreating, or in curing, alleviating, relieving or improving a subjectwith a disorder beyond that expected in the absence of such treatment.This may include a particular concentration or range of concentrationsof peptide in the peptide solution or hydrogel and additionally, or inthe alternative, a particular volume or range of volumes of the peptidesolution or hydrogel. The method of facilitating may comprise providinginstructions to prepare at least one of the effective amount and theeffective concentration.

The dosage, for example, volume or concentration, administered (forexample, applied or injected) may vary depending upon the form of thepeptide (for example, in a peptide solution, hydrogel, or in a driedform, such as a lyophilized form) and the route of administrationutilized. The exact formulation, route of administration, volume, andconcentration can be chosen in view of the subject's condition and inview of the particular target area or location that the peptidesolution, hydrogel, or other form of peptide will be administered. Loweror higher doses than those recited herein may be used or required.Specific dosage and treatment regimens for any particular subject maydepend upon a variety of factors, which may include the specific peptideor peptides employed, the dimension of the biological vessel that isbeing treated or occluded, the desired thickness of the resultinghydrogel that may be positioned in the desired target area, and thelength of time of treatment. Other factors that may affect the specificdosage and treatment regimens include age, body weight, general healthstatus, sex, time of administration, rate of degradation, the severityand course of the disease, condition or symptoms, and the judgment ofthe treating physician. In certain embodiments, the peptide solution maybe administered in a single dose. In other embodiments, the peptidesolution may be administered in more than one dose, or multiple doses.

An effective amount and an effective concentration of the peptidesolution may be selected to at least partially obstruct or block abiological vessel. In some embodiments, at least one of the effectiveamount and the effective concentration may be based in part on adiameter of the target area of the biological vessel. In otherembodiments, at least one of the effective amount and the effectiveconcentration is based in part on the flow rate of the blood in thebiological vessel. In other embodiments, at least one of the effectiveamount and the effective concentration may be based in part on a bloodpressure of the blood in the biological vessel. In still otherembodiments, at least one of the effective amount and the effectiveconcentration may be based in part on an average diameter of a red bloodcell of the subject.

In yet other embodiments, at least one of the effective amount and theeffective concentration may be based in part on at least one of thediameter of the target area of the biological vessel, the flow rate ofblood in the biological vessel, the blood pressure of the blood in thebiological vessel, and the average diameter of a red blood cell of thesubject.

The at least one of the effective amount and the effective concentrationmay be based in part on providing nanofibers of a hydrogel having anaverage pore size that is less than an average diameter of a red bloodcell of the subject. This may comprise collecting a sample of blood fromthe subject to determine the average red blood cell diameter to providefor the at least one of the effective amount and the effectiveconcentration.

The effective amount may be, as described herein, an amount that mayprovide for a desired blockage in a biological vessel. Variousproperties of the biological vessel may contribute to the selection ordetermination of the effective amount including at least one of thediameter of the target area of the biological vessel, the flow rate ofblood in the biological vessel, the blood pressure of the blood in thebiological vessel, and the average diameter of a red blood cell of thesubject.

The effective amount may include volumes of from about 0.1 milliliters(mL) to about 100 mL of a peptide solution. The effective amount mayinclude volumes of from about 0.1 mL to about 10 mL of a peptidesolution. In certain embodiments, the effective amount may be about 0.5mL. In other embodiments, the effective amount may be about 1.0 mL. Inyet other embodiments, the effective amount may be about 1.5 mL. Instill yet other embodiments, the effective amount may be about 2.0 mL.In some other embodiments, the effective amount may be about 3.0 mL.

In some embodiments, a more effective blockage may be achieved with agreater volume of peptide solution administered. This may allow a longeror thicker hydrogel to form within the biological vessel, allowing amore secure position of the hydrogel in the target area. It is possiblethat if a high enough volume is not selected, the hydrogel may not beeffective in maintaining a blockage in the target area for the desiredperiod of time. This may also be influenced based on the blood flow rateor blood pressure in the vessel.

The effective concentration may be, as described herein, an amount thatmay provide for a desired blockage in a biological vessel. Variousproperties of the biological vessel may contribute to the selection ordetermination of the effective concentration including at least one ofthe diameter of the target area of the biological vessel, the flow rateof blood in the biological vessel, the blood pressure of the blood inthe biological vessel, and the average diameter of a red blood cell ofthe subject.

The effective concentration may include peptide concentrations in thesolution in a range of about 0.1 weight per volume (w/v) percent toabout 10 w/v percent. The effective concentration may include peptideconcentrations in the solution in a range of about 0.1 w/v percent toabout 3.5 w/v percent. In certain embodiments, the effectiveconcentration may be about 1 w/v percent. In other embodiments, theeffective concentration may be about 2.5 w/v percent. In yet otherembodiments, the effective concentration may be about 3.0 w/v percent.

In certain embodiments, a peptide solution having a higher concentrationof peptide may provide for a more effective hydrogel that has theability to stay in place and provide effective blockage of thebiological vessel. For purposes of delivering the peptide solution,higher concentrations of peptide solutions may become too viscous toallow for effective and selective administration of the solution. It ispossible that if a high enough concentration is not selected, thehydrogel may not be effective in maintaining a blockage in the targetarea for the desired period of time. This may also be influenced basedon the blood flow rate or blood pressure in the vessel.

The effective concentration may be selected to provide for a solutionthat may be administered by injection or other means using a particulardiameter or gauge catheter or needle.

Methods of the disclosure contemplate single as well as multipleadministrations of a therapeutically effective amount of the peptides,peptide solutions, and hydrogels as described herein. Peptides asdescribed herein may be administered at regular intervals, depending onthe nature, severity and extent of the subject's condition. In someembodiments, a peptide, peptide solution, or hydrogel may beadministered in a single administration. In some embodiments, a peptide,peptide solution, or hydrogel described herein is administered inmultiple administrations. In some embodiments, a therapeuticallyeffective amount of a peptide, peptide solution, or hydrogel may beadministered periodically at regular intervals. The regular intervalsselected may be based on any one or more of the initial peptideconcentration of the solution administered, the amount administered, andthe degradation rate of the hydrogel formed. For example, after aninitial administration, a follow-on administration may occur after, forexample, two weeks, four weeks, six weeks, or eight weeks. The follow-onadministration may comprise administration of a solution having the sameconcentration of peptide and volume as the initial administration, ormay comprise administration of a solution of lesser or greatconcentration of peptide and volume. The selection of the appropriatefollow-on administration of peptide solution may be based on imaging thetarget area and the area surrounding the target area and ascertainingthe needs based on the condition of the subject. The pre-determinedintervals may be the same for each follow-on administration, or they maybe different. In some embodiments, a peptide, peptide solution, orhydrogel may be administered chronically at pre-determined intervals tomaintain at least a partial blockage of a biological vessel in a subjectover the life of the subject. The pre-determined intervals may be thesame for each follow-on administration, or they may be different. Thismay be dependent on whether the hydrogel formed from the previousadministration is partially or totally disrupted or degraded. Thefollow-on administration may comprise administration of a solutionhaving the same concentration of peptide and volume as the initialadministration, or may comprise administration of a solution of lesseror great concentration of peptide and volume. The selection of theappropriate follow-on administration of peptide solution may be based onimaging the target area and the area surrounding the target area andascertaining the needs based on the condition of the subject.

The self-assembling peptides of the present disclosure, such as RADA16(SEQ ID NO: 7), may be peptide sequences that lack a distinctphysiologically or biologically active motif or sequence, and thereforemay not impair intrinsic cell function. Physiologically active motifsmay control numerous intracellular phenomena such as transcription, andthe presence of physiologically active motifs may lead tophosphorylation of intracytoplasmic or cell surface proteins by enzymesthat recognize the motifs. When a physiologically active motif ispresent in a peptide tissue occluding agent, transcription of proteinswith various functions may be activated or suppressed. Theself-assembling peptides, of the present disclosure may lack suchphysiologically active motifs and therefore do not carry this risk.

A sugar may be added to the self-assembling peptide solution to improvethe osmotic pressure of the solution from hypotonicity to isotonicitywithout reducing the tissue occluding effect, thereby allowing thebiological safety to be increased. In certain examples, the sugar may besucrose or glucose.

In certain embodiments, the peptide length may be more than 12 aminoacids and preferably at least 16 residues. Very long peptides, forexample, of about 200 amino acids, may encounter problems due toinsolubility and intramolecular interactions which destabilize membraneformation, but may also be contemplated herein. Furthermore, peptideswith a large amount of hydrophobic residues may have insolubilityproblems. The optimal lengths for membrane formation may vary with theamino acid composition.

An additional stabilization factor is that complementary peptidesmaintain a constant distance between the peptide backbones. Peptideswhich can maintain a constant distance upon pairing are referred toherein as structurally compatible. The interpeptide distance can becalculated for each ionized or hydrogen bonding pair by taking the sumof the number of unbranched atoms on the side-chains of each amino acidin the pair. For example, lysine has 5 and glutamic acid has 4unbranched atoms on its side-chains, respectively.

Examples of peptides that may form membranes in homogeneous mixtures areshown in Table 1. These examples illustrate some of the variety of aminoacid arrangement and composition of membrane-forming peptides.

TABLE 1  Potential membrane-forming peptides Name Sequence (N→C) IEIK13IEIKIEIKIEIKI (SEQ ID NO: 8) IEIK17 IEIKIEIKIEIKIEIKI (SEQ ID NO: 9)KAKA16 KAKAKAKAKAKAKAKA (SEQ ID NO: 12) KAKA5 KAKAK (SEQ ID NO: 13)KAE16 AKAKAEAEAKAKAEAE (SEQ ID NO: 14) AKE16AKAEAKAEAKAEAKAE (SEQ ID NO: 15) EKA16 EAKAEAKAEAKAEAKA (SEQ ID NO: 11)EAK8 AEAEAKAK (SEQ ID NO: 16) EAK12 AEAKAEAEAKAK (SEQ ID NO: 17) KEA16KAEAKAEAKAEAKAEA (SEQ ID NO: 18) AEK16 AEAKAEAKAEAKAEAK (SEQ ID NO: 19)ARD8 ARARADAD (SEQ ID NO: 20) DAR16 ADADARARADADARAR (SEQ ID NO: 21)RAD16 ARADARADARADARAD (SEQ ID NO: 22) DRA16DARADARADARADARA (SEQ ID NO: 23) RADA16 RADARADARADARADA (SEQ ID NO: 7)ADR16 ADARADARADARADAR (SEQ ID NO: 24) ARA16ARARADADARARADAD (SEQ ID NO: 25) ARDAKE16ARADAKAEARADAKAE (SEQ ID NO: 26) AKEW16 AKAEARADAKAEARAD (SEQ ID NO: 27)ARKADE16 ARAKADAEARAKADAE (SEQ ID NO: 28) AKRAED16AKARAEADAKARADAE (SEQ ID NO: 29) AQ16 AQAQAQAQAQAQAQAQ (SEQ ID NO: 30)VQ16 VQVQVQVQVQVQVQVQ (SEQ ID NO: 31) YQ16YQYQYQYQYQYQYQYQ (SEQ ID NO: 32) HQ16 HQHQHQHQHQHQHQHQ (SEQ ID NO: 33)AN16 ANANANANANANANAN (SEQ ID NO: 34) VN16VNVNVNVNVNVNVNVN (SEQ ID NO: 35) YN16 YNYNYNYNYNYNYNYN (SEQ ID NO: 36)HN16 HNHNHNHNHNHNHNHN (SEQ ID NO: 37) ANQ16ANAQANAQANAQANAQ (SEQ ID NO: 38) AQN16 AQANAQANAQANAQAN (SEQ ID NO: 39)VNQ16 VNVQVNVQVNVQVNVQ (SEQ ID NO: 40) VQK16VQVNVQVNVQVNVQVN (SEQ ID NO: 41) YNQ16 YNYQYNYQYNYQYNYQ (SEQ ID NO: 42)YQN16 YQYNYQYNYQYNYQYN (SEQ ID NO: 43) HNQ16HNHQHNHQHNHQHNHQ (SEQ ID NO: 44) HQN16 HQHNHQHNHQHNHQHN (SEQ ID NO: 45)AKQD18 AKAQADAKAQADAKAQAD (SEQ ID NO: 46) VKQ18VKVQVDVKVQVDVKVQVD (SEQ ID NO: 47) YKQ18YKYQYDYKYQYDYKYQYD (SEQ ID NO: 48) HKQ18HKHQHDHKHQHDHKHQHD (SEQ ID NO: 49) β-Amyloid DAEFRHDSGYEVHHQKLVFFAEDVGSNK  (1-28) (SEQ ID NO: 50) β-Amyloid GSNKGAIIGLM (SEQ ID NO: 51) (25-35) Substance PRPKQQFGLM (SEQ ID NO: 52) Spantide (D)RPKPQQ(D)WF(D)WLL * (SEQ ID NO: 53) * (D) in Spantide is a D amino acid incorporated intothe peptide

The criteria of amphiphilic sequence, length, complementarity andstructural compatibility apply to heterogeneous mixtures of peptides.For example, two different peptides may be used to form the membranes:peptide A,Val-Arg-Val-Arg-Val-Asp-Val-Asp-Val-Arg-Val-Arg-Val-Asp-Val-Asp(VRVRVDVDVRVRVDVD) (SEQ ID NO: 54), as shown in the appended sequencelisting), has Arg and Asp as the hydrophilic residues and peptide B,Ala-Asp-Ala-Asp-Ala-Lys-Ala-Lys-Ala-Asp-Ala-Asp-Ala-Lys-Ala-Lys(ADADAKAKADADAKAK) (SEQ ID NO: 55), has Lys and Asp. Peptides A and Bare complementary; the Arg on A can form an ionized pair with the Asp onB and the Asp on A can form an ionized pair with the Lys on B. Acalculation of the interpeptide distances in such pairs, however, showsthat the two peptides are not structurally compatible. Using aconversion factor of 3 Angstroms per atom, the difference ininterpeptide distance between the two pairs would be 3 Angstroms. It isestimated that a variation in interpeptide distance of more than 3-4Angstroms would destabilize intermolecular interactions leading tomembrane formation. Thus, in a heterogeneous mixture of peptides A andB, membranes would likely form, but they would be homogeneously composedof either peptide A or B.

Membranes and hydrogels may also be formed of heterogeneous mixtures ofpeptides, each of which alone would not form membranes, if they arecomplementary and structurally compatible to each other. For example,mixtures of (Lys-Ala-Lys-Ala)₄ (KAKA)₄ (SEQ ID NO: 12) and(Glu-Ala-Glu-Ala)₄ (EAEA)₄ (SEQ ID NO: 56) or of (Lys-Ala-Lys-Ala)₄(KAKA)₄ (SEQ ID NO: 12) and (Ala-Asp-Ala-Asp)₄ (ADAD)₄ (SEQ ID NO: 57)would be expected to form membranes, but not any of these peptides alonedue to lack of complementarity.

Peptides, which are not perfectly complementary or structurallycompatible, can be thought of as containing mismatches analogous tomismatched base pairs in the hybridization of nucleic acids. Peptidescontaining mismatches can form membranes if the disruptive force of themismatched pair is dominated by the overall stability of theinterpeptide interaction. Functionally, such peptides can also beconsidered as complementary or structurally compatible. For example, amismatched amino acid pair may be tolerated if it is surrounded byseveral perfectly matched pairs on each side. Mismatched peptides can betested for ability to self-assemble into macroscopic membranes using themethods described herein.

The peptides can be chemically synthesized or they can be purified fromnatural and recombinant sources. Using chemically synthesized peptidesmay allow the peptide solutions to be deficient in unidentifiedcomponents such as unidentified components derived from theextracellular matrix of another animal. This property therefore mayeliminate concerns of infection, including risk of viral infectioncompared to conventional tissue-derived biomaterials. This may eliminateconcerns of infection including infections such as bovine spongiformencephalopathy (BSE), making the peptide highly safe for medical use.

The initial concentration of the peptide may be a factor in the size andthickness of the membrane or hydrogel formed. In general, it may be thecase that the higher the peptide concentration, the higher the extent ofmembrane formation. Membranes or hydrogels may form from initial peptideconcentrations as low as about 0.5 mM or about 1 mg/ml (about 0.1 w/vpercent). However, membranes or hydrogels formed at higher initialpeptide concentrations (about 10 mg/ml (about 1 w/v percent)) may bethicker and thus, likely to be stronger. It may be preferable whenproducing the membranes or hydrogels to add peptide to a salt solutionor a physiological condition, rather than to add salt to a peptidesolution.

Formation of the membranes or hydrogels may be very fast, on the orderof a few minutes. The formation of the membranes or hydrogels may forminstantaneously upon application or injection to a desired area. Theformation of the membranes or hydrogels may occur within one to twominutes of application or injection. In other examples, the formation ofthe membranes or hydrogels may occur within four minutes of applicationor injection. In certain embodiments the time it takes to form themembranes or hydrogels may be based at least in part on one or more ofthe concentration of the peptide solution, the volume of peptidesolution applied, and the conditions at the area of application orinjection (for example, the concentration of monovalent metal cations atthe area of application, the blood flow rate, the blood pressure, andthe diameter of the biological vessel).

The formation of the membranes or hydrogels may be irreversible. Theprocess may be unaffected by pH of less than or equal to 12 (thepeptides tend to precipitate out at pH above 12), and by temperature.The membranes or hydrogels may form at temperatures in the range of 4 to90 degrees Celsius.

The membranes or hydrogels may remain in position at the target area fora period of time sufficient to provide a desired effect using themethods and kits of the present disclosure. The desired effect may be toreduce or prevent flow of a fluid through a biological pathway orchannel. The desired effect may be a blockage, lodging, occlusion, orembolism in one or more biological pathways or channels. The desiredeffect may be to purposely create such a blockage, lodging or occlusionin order to deprive tumors or other pathological processes of theirblood supply (perfusion).

The desired effect using the methods and kits of the present disclosuremay be to treat disorders, malformations, or congenital ailments inbiological vessels. The desired effect may be to treat one or more ofpatent ductus arteriosus (PDA), major aortopulmonary collateral artery(MAPCA), recurrent hemotysis, arteriovenous malformations, cerebralaneurysms, gastrointestinal bleeding, epistaxis, post-partum hemorrhage,surgical hemorrhage, and uterine fibroids. The desired effect mayinclude providing at least a partial blockage to produce cell necrosisor to reduce or eliminate cancerous cells.

The period of time that the membranes or hydrogels may remain at thedesired area may be for about 10 minutes. In certain examples, it mayremain at the desired area for about 35 minutes. In certain furtherexamples, it may remain at the desired area for several days, up to twoweeks. In other examples, it may remain at the desired areaindefinitely. In other examples, it may remain at the desired area for alonger period of time, until it is naturally degraded or intentionallyremoved. If the hydrogel naturally degrades over a period of time,subsequent application or injection of the hydrogel to the same ordifferent location in the biological vessel, or another biologicalvessel may be performed.

In certain embodiments, the self-assembling peptide may be prepared withone or more components that may provide for enhanced effectiveness ofthe self-assembling peptide or may provide another action, treatment,therapy, or otherwise interact with one or more components of thesubject. For example, additional peptides comprising one or morebiologically or physiologically active amino acid sequences or motifsmay be included as one of the components along with the self-assemblingpeptide. Other components may include biologically active compounds suchas a drug or other treatment that may provide some benefit to thesubject. For example, a cancer treating drug or anticancer drug may beadministered with the self-assembling peptide, or may be administeredseparately.

The peptide, peptide solution, or hydrogel may comprise small moleculardrugs to treat the subject or to prevent hemolysis, inflammation, andinfection. The small molecular drugs may be selected from the groupconsisting of glucose, saccharose, purified saccharose, lactose,maltose, trehalose, destran, iodine, lysozyme chloride,dimethylisoprpylazulene, tretinoin tocoferil, povidone iodine,alprostadil alfadex, anise alcohol, isoamyl salicylate,α,α-dimethylphenylethyl alcohol, bacdanol, helional, sulfazin silver,bucladesine sodium, alprostadil alfadex, gentamycin sulfate,tetracycline hydrochloride, sodium fusidate, mupirocin calcium hydrateand isoamyl benzoate. Other small molecular drugs may be contemplated.Protein-based drugs may be included as a component to be administered,and may include erythropoietin, tissue type plasminogen activator,synthetic hemoglobin and insulin.

A component may be included to protect the peptide solution againstrapid or immediate formation into a hydrogel. This may include anencapsulated delivery systems that may degrade over time to allow acontrolled time release of the peptide solution into the target area toform the hydrogel over time a desired, predetermined period of time.Biodegradable, biocompatible polymers may be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid.

Any of the components described herein may be included in the peptidesolution or may be administered separate from the peptide solution.Additionally, any of the methods and methods of facilitating providedherein may be performed by one or more parties.

A peptide, peptide solution, or hydrogel of the disclosure may beprovided in a kit. Instructions for administering the solution to abiological vessel in a subject may also be provided in the kit. Thepeptide solution may comprise an amphiphilic peptide comprising at least12 amino acids that alternate between a hydrophobic amino acid and ahydrophilic amino acid in an effective amount and in an effectiveconcentration to form a hydrogel under physiological conditions to allowat least partial blockage of a biological vessel. The instructions foradministering the solution may comprise methods for administering thepeptide, peptide solution, or hydrogel provided herein, for example, bya route of administration described herein, at a dose, volume orconcentration, or administration schedule.

The kit may also comprise informational material. The informationalmaterial may be descriptive, instructional, marketing or other materialthat relates to the methods described herein. In one embodiment, theinformational material may include information about production of thepeptide, peptide solution, or hydrogel disclosed herein, physicalproperties of the peptide, peptide solution or hydrogel, concentration,volume, size, dimensions, date of expiration, and batch or productionsite.

The kit may also optionally include a device or materials to allow foradministration of the peptide or peptide solution to the desired area.For example, a syringe, pipette, catheter, or other needle-based devicemay be included in the kit. Additionally, or alternatively, the kit mayinclude a guidewire or other accompanying equipment to provide selectiveadministration of the peptide solution to the target area.

The kit may comprise in addition to or in the alternative, othercomponents or ingredients, such as components that may aid in contrastimaging. For example, the kit may comprise a contrast agent. Thecontrast agent may provide a visual image during use of X-raytechniques, such as fluoroscopy or angiography. A nonionic radiopaquecontrast media may be included, such as, for example, a water-solubleiodine based solution. The water-soluble iodine based solution may beiopamidol. Instructions may be provided in the kit to combine asufficient quantity or volume of the contrast agent in order toadequately do at least one of: identify the target area, introduce acatheter or other administration device, position an end of the catheterin the target area, administer the peptide solution, remove the catheteror other administration device, and observe the biological vessel afterremoving the catheter. The use of the contrast agent may allowvisualization of the area to which the peptide, peptide solution orhydrogel is administered. Instructions may be provided for diluting thepeptide solution to administer an effective concentration of thesolution to the biological vessel. Instructions may further be providedfor determining at least one of the effective concentration of thesolution and the effective amount of the solution to the biologicalvessel. This may be based on various parameters discussed herein, andmay include the diameter of the biological vessel at the target area.

Other components or ingredients may be included in the kit, in the sameor different compositions or containers than the peptide, peptidesolutions, or hydrogel. The one or more components that may includecomponents that may provide for enhanced effectiveness of theself-assembling peptide or may provide another action, treatment,therapy, or otherwise interact with one or more components of thesubject. For example, additional peptides comprising one or morebiologically or physiologically active sequences or motifs may beincluded as one of the components along with the self-assemblingpeptide. Other components may include biologically active compounds suchas a drug or other treatment that may provide some benefit to thesubject. For example, a cancer treating drug or anticancer drug may beadministered with the self-assembling peptide, or may be administeredseparately. The peptide, peptide solution, or hydrogel may comprisesmall molecular drugs to treat the subject or to prevent hemolysis,inflammation, and infection, as disclosed herein. A sugar solution suchas a sucrose solution may be provided with the kit. The sucrose solutionmay be a 20% sucrose solution.

Other components which are disclosed herein may also be included in thekit.

In some embodiments, a component of the kit is stored in a sealed vial,for example, with a rubber or silicone closure (for example, apolybutadiene or polyisoprene closure). In some embodiments, a componentof the kit is stored under inert conditions (for example, under nitrogenor another inert gas such as argon). In some embodiments, a component ofthe kit is stored under anhydrous conditions (for example, with adesiccant). In some embodiments, a component of the kit is stored in alight blocking container such as an amber vial.

As part of the kit or separate from a kit, syringes or pipettes may bepre-filled with a peptide, peptide solution, or hydrogel as disclosedherein. Methods to instruct a user to supply a self-assembling peptidesolution to a syringe or pipette, with or without the use of otherdevices, and administering it to the target area through the syringe orpipette, with or without the use of other devices, is provided. Otherdevices may include, for example, a catheter with or without aguidewire.

In some embodiments of the disclosure, the self-assembling peptides maybe used as a coating on a device or an instrument such as a stent orcatheter, to suppress body fluid leakage. The self-assembling peptidesmay also be incorporated or secured to a support, such as gauze or abandage, or a lining, that may provide a therapeutic effect to asubject, or that may be applied within a biological vessel. Theself-assembling peptides may also be soaked into a sponge for use.

In alternative embodiments, an atomizing sprayer filled with a powder orsolution of the self-assembling peptides may be prepared. When such aspray is used for spraying onto an affected area, the pH and saltconcentration increase upon contact with the body causing gelling.

Modification of the membranes may give them additional properties. Forexample, the membranes may be further strengthened by cross-linking thepeptides after membrane formation by standard methods. Collagen may becombined with the peptides to produce membranes more suitable for use asartificial skin; the collagen may be stabilized from proteolyticdigestion within the membrane. Furthermore, combining phospholipids withthe peptides may produce vesicles.

The membranes may also be useful for culturing cell monolayers. Cellsprefer to adhere to non-uniform, charged surfaces. The charged residuesand conformation of the proteinaceous membranes promote cell adhesionand migration. The addition of growth factors, such as fibroblast growthfactor, to the peptide membrane can further improve attachment, cellgrowth and neurite outgrowth.

The function and advantage of these and other embodiments of the methodsand kits disclosed herein will be more fully understood from the examplebelow. The following example is intended to illustrate the benefits ofthe disclosed treatment approach, but do not exemplify the full scopethereof.

EXAMPLES Example 1

Tests were performed on a rat using a 3% (weight per volume (w/v))PuraMatrix™ solution, a peptide solution comprisingAc-RADARADARADARADA-NH₂(Ac-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-NH₂)(SEQ ID NO: 58) in water. An 18 gauge needle was used to inject 1milliliter (mL) into a rat portal vein. Hematoxylin-Eosin (HE) dye wasused to implement a histopathological evaluation. It was confirmed thatan embolism developed in the portal vein using the peptide solution. Asshown in FIG. 1, the peptide solution appears to have developed into ahydrogel 2 and resides in the rat portal vein. A red blood cell 4 isalso shown.

Example 2

Tests were performed in two beagles using a 2.5% PuraMatrix™ solution, apeptide solution comprising Ac-RADARADARADARADA-NH₂(Ac-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-NH₂)(SEQ ID NO: 58) in water. These tests were performed to confirm theeffectiveness of beagle hepatic artery embolism and hepatic cellnecrosis using the 2.5% peptide solution. The peptide solution includediopamidol at a concentration of 612.4 mg/mL. Iopamidol is a nonionicradiopaque contrast agent.

Under X-ray imaging of a beagle under full anesthesia, a microcatheter(Terumo, minimum inner diameter of 0.50 mm) was inserted by way of thecarotid artery into the hepatic artery. Hepatic artery contrast imagingwas used to confirm that the hepatic artery was operational. A 2 mLvolume of the 2.5% peptide solution (with Iopamidol) was injected.

Hepatic artery contrast imaging was used to confirm the hepatic arterypeptide solution embolism effect during surgery. FIG. 2 displays acontrast image of a normal hepatic artery of the beagle, while FIG. 3shows the peptide solution injection. FIG. 4 shows a hepatic arterycontrast image after a peptide solution injection in which back flow ofthe contrast image was confirmed. The presence of the peptide solutionis evidenced by the darker regions of the image.

After two weeks of monitoring elapsed, hepatic artery contrast imagingwas used to confirm the embolism effect. FIG. 5 shows a hepatic arterycontrast image two weeks after a peptide solution injection.

Subsequently, the liver was extracted. Hematoxylin-Eosin (HE) dye wasused to histopathologically confirm the peptide solution embolism andhepatic impairment. As shown in FIGS. 6A-6C, the peptide solution can beseen in each of these images of the hepatic artery as the darkened areasof the images. FIG. 7 shows a hepatic cell necrosis image at an embolismlocation, where all cells appear to have at least some level ofnecrosis.

The results show that injection of the peptide solution using amicrocatheter may be accomplished. The peptide gel may be visible usingX-ray imaging. The hepatic artery embolism effect can be seen duringsurgery and two weeks after surgery. Additionally, the hepatic arteryembolism effect and hepatic cell necrosis effect using the peptidesolution occurred and was confirmed histopathologically.

Example 3

PuraMatrix™, a peptide solution comprising Ac-RADARADARADARADA-NH₂(Ac-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-Arg-Ala-Asp-Ala-NH₂)(SEQ ID NO: 58) in water was used as an embolic agent in a porcine modelthrough angiography, gross necropsy assessment, and histopathologyassessment. One female Yorkshire cross swine was tested. The weight ofthe swine at the time of testing was 46.5 kg. Feed and water wereprovided per standard operating procedures. There were no contaminantsin the food or water that were expected to interfere with the conduct orresults of the study. The swine was acquired from a test facilityapproved animal supplier. The Swine participated in an incoming physicalexam, and after a period of acclimation was again examined. The swinewas fasted a minimum of 12 hours prior to the procedures. The animalswere sedated and anesthetized by an intramuscular or subcutaneousinjection of Telazol (2-10 mg/kg) and Xylazine (0.5=5.0 mg/kg). Propofol(to effect) was given to aid in sedation. An endotracheal tube was usedto ensure proper ventilation and the animals were maintained undergeneral anesthesia with inhalant isofluorante (0.1 to 5.0%). Heparin(50-300 units/kg, IV) was administered throughout the procedure.

A 2.5% test solution of the peptide solution was used. Approximately 800microliters of the peptide solution was placed in an eppendorf tube.Approximately 200 microliters of Isovue-370 (Iopamidol) contrast agentwas added. The liquids were mixed slowly so as to not create airbubbles.

On the day of testing, the swine was 2 months, 25 days old. The swinewas sedated and prepared for surgery. The femoral artery was accessedand an introducer was placed. A guidewire was advanced to the selectedrenal artery. A catheter was advanced to the selected renal artery.Angiography was used to visualize the location within the artery. Thepeptide solution was injected to the desired location until the arterywas occluded. This procedure was repeated in the hepatic and splenicarteries. Angiography was used throughout the procedure to visualize thevessels and devices throughout testing. FIGS. 8 and 9 are representativeexamples of a vessel before (FIG. 6) and after (FIG. 9) embolization.There were no adverse events reported throughout the testing.

A summary of the data can be found in Table 2 below.

TABLE 2 Approximate Test Embolization Volume Number Site Time PlacedComments 1 Left Start 13:55 1.5 mL Successful Kidney embolization Renalimmediately following Artery injection of peptide solution/Isovue 2Right Start 14:12 1.5 mL Successful Kidney embolization Renalimmediately following Artery injection. Slight flow reestablished at14:18. Additional PuraMatrix ™/ Iopamidol placed at 14:27. Angiogramshowed full occlusion. 3 Hepatic Start 14:58 2.0 mL Successful Arteryembolization immediately following injection. At 15:08, the arteryremained occluded. At 15:32, the artery remained occluded. 4 SplenicStart 15:17 3.0 mL Vessel was completed Artery occluded at 15:21.

As shown in Table 2, injection into the left kidney renal artery wassuccessful, immediately following injection. Successful embolization ofthe right kidney renal artery was successful immediately followinginjection, however, a slight flow was reestablished 6 minutes after theinitial injection. An additional injection was made 15 minutes after theinitial injection, and a full occlusion was obtained.

Successful embolization of the hepatic artery was also obtainedimmediately following injection. The artery remained occluded after 10minutes and 34 minutes. Successful embolization of the splenic arterywas also obtained after four minutes.

The description and figures provided are for example only and are notintended to be limiting. While exemplary embodiments of the disclosurehave been disclosed many modifications, additions, and deletions may bemade therein without departing from the spirit and scope of thedisclosure and its equivalents, as set forth in the following claims.

Those skilled in the art would readily appreciate that the variousconfigurations described herein are meant to be exemplary and thatactual configurations will depend upon the specific application forwhich the system and methods of the present disclosure are used. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, many equivalents to the specificembodiments described herein.

Further, it is to be appreciated various alterations, modifications, andimprovements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe disclosure. Accordingly, the foregoing description and drawings areby way of example only. Further, the depictions in the drawings do notlimit the disclosures to the particularly illustrated representations.

As used herein, the terms “comprising,” “including,” “carrying,”“having,” “containing,” and “involving,” whether in the writtendescription or the claims and the like, are open-ended terms, i.e., tomean “including but not limited to.” Thus, the use of such terms ismeant to encompass the items listed thereafter, and equivalents thereof,as well as additional items. Only the transitional phrases “consistingof” and “consisting essentially of,” are closed or semi-closedtransitional phrases, respectively, with respect to the claims Use ofordinal terms such as “first,” “second,” “third,” and the like in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

1. A method of blocking a biological vessel in a subject comprising:introducing a catheter into a biological vessel; positioning an end ofthe catheter in a target area of the biological vessel in which at leasta partial obstruction is desired; administering through the catheter asolution comprising an amphiphilic peptide comprising at least 12 aminoacids that alternate between a hydrophobic amino acid and a hydrophilicamino acid in an effective amount and in an effective concentration toform a hydrogel under physiological conditions to allow at least partialblockage of the biological vessel; removing the catheter from thebiological vessel with the at least partial obstruction in place.
 2. Themethod of claim 1, wherein the peptide solution comprises a contrastagent.
 3. The method of claim 2, further comprising visualizing a regioncomprising at least a portion of the biological vessel.
 4. The method ofclaim 3, wherein visualizing the region comprising at least a portion ofthe biological vessel comprises visualizing the region during at leastone of: identifying the target area of the biological vessel;introducing the catheter; positioning the end of the catheter in thetarget area; administering the solution; removing the catheter; andvisualizing the biological vessel after removing the catheter.
 5. Themethod of claim 4, wherein visualizing the region comprises imagingusing X-ray radiography.
 6. The method of claim 3, wherein visualizingthe region provides for selective administration of the solution to thebiological vessel.
 7. The method of claim 3, further comprisingvisualizing the region in a time period about two weeks subsequent theadministration.
 8. The method of claim 1, wherein at least one of theeffective amount and the effective concentration is based in part on adiameter of the target area of the biological vessel.
 9. The method ofclaim 1, wherein at least one of the effective amount and the effectiveconcentration is based in part on the flow rate of the blood in thebiological vessel.
 10. The method of claim 1, wherein at least one ofthe effective amount and the effective concentration is based in part onproviding nanofibers of the hydrogel having an average pore size that isless than an average diameter of a red blood cell of the subject. 11.The method of claim 1, wherein the concentration effective to allow atleast partial blockage of the biological vessel comprises aconcentration in a range of about 0.1 weight per volume (w/v) percent toabout 3 w/v percent peptide.
 12. The method of claim 1, wherein theamount effective to allow at least partial blockage of the biologicalvessel comprises a volume in a range of about 0.1 mL to about 5 mL. 13.The method of claim 1, further comprising monitoring the areasurrounding the at least partial blockage to determine an effectivenessof the at least partial obstruction.
 14. The method of claim 1, whereinthe formed blockage is used in the treatment of disorders,malformations, or congenital ailments in biological vessels.
 15. Themethod of claim 14, wherein the formed blockage is used in the treatmentof one of patent ductus arteriosus (PDA) and major aortopulmonarycollateral artery (MAPCA).
 16. The method of claim 14, wherein theformed blockage is used in the treatment of a disorder, malformation, orcongenital ailment selected from the group consisting of recurrenthemotysis, arteriovenous malformations, cerebral aneurysms,gastrointestinal bleeding, epistaxis, post-partum hemorrhage, surgicalhemorrhage, and uterine fibroids.
 17. The method of claim 1, wherein theformed blockage is used in the reduction of cancerous cells.
 18. Themethod of claim 1, wherein the peptide solution is substantially free ofcells.
 19. The method of claim 1, wherein the peptide solution issubstantially free of drugs.
 20. The method of claim 1, wherein thesubject is a mammal.
 21. The method of claim 20, wherein the subject ishuman.
 22. The method of claim 1, wherein administering the solutioncomprises administering the solution in a single dose.
 23. The method ofclaim 1, wherein administering the solution comprises administering thesolution in at least two doses.
 24. The method of claim 1, wherein thepeptide has an amino acid sequence of one of RADARADARADARADA (SEQ IDNO: 7), IEIKIEIKIEIKI (SEQ ID NO: 8), and IEIKIEIKIEIKIEIKI (SEQ ID NO:9).
 25. The method of claim 1, further comprising evaluating the subjectto determine a need for blocking a biological vessel and preparing thepeptide solution.
 26. The method of claim 25, wherein preparing thepeptide solution comprises adding a contrast agent to a preliminarysolution comprising peptides.
 27. The method of claim 1, wherein thesolution is administered to allow complete blockage of the biologicalvessel.
 28. The method of claim 1, further comprising introducing aguidewire into the biological vessel prior to introducing the catheter.29. A kit for blocking a biological vessel in a subject comprising: asolution comprising an amphiphilic peptide comprising at least 12 aminoacids that alternate between a hydrophobic amino acid and a hydrophilicamino acid in an effective amount and in an effective concentration toform a hydrogel under physiological conditions to allow at least partialblockage of the biological vessel; and instructions for administeringthe solution to the biological vessel in the subject.
 30. The kit ofclaim 29, further comprising a catheter to introduce the solution intothe biological vessel of the subject.
 31. The kit of claim 29, furthercomprising instructions for adding contrast agent to the solution in anappropriate amount to visualize administering the solution.
 32. The kitof claim 31, further comprising at least one of a contrast agent and asucrose solution.
 33. The kit of claim 29, further comprisinginstructions for diluting the solution to administer an effectiveconcentration of the solution to the biological vessel in the subject.34. The kit of claim 33, further comprising instructions for determiningthe effective concentration of the solution to the biological vessel inthe subject based on the diameter of the biological vessel at a targetarea.
 35. A method of facilitating blocking a biological vessel in asubject comprising: providing a solution comprising an amphiphilicpeptide comprising at least 12 amino acids that alternate between ahydrophobic amino acid and a hydrophilic amino acid in an effectiveamount and in an effective concentration to form a hydrogel underphysiological conditions to allow at least partial blockage of thebiological vessel; and providing instructions for administering thesolution to a target area of the biological vessel through introductionof the solution to a catheter positioned in the biological vessel. 36.The method of claim 35, further comprising providing instructions to adda contrast agent to the solution.
 37. The method of claim 36, furthercomprising providing instructions to visualize a region comprising atleast a portion of the biological vessel.
 38. The method of claim 37,wherein providing instructions to visualize the region comprising atleast a portion of the biological vessel comprises providing instructionto visualize the region during at least one of: identifying the targetarea of the biological vessel; introducing a catheter; positioning anend of the catheter in the target area; administering the solution;removing the catheter from the biological vessel with the at leastpartial blockage in place; and visualizing the biological vessel afterremoving the catheter.
 39. The method of claim 38, wherein providinginstructions to visualize the region comprises imaging using X-rayradiography.
 40. The method of claim 37, further comprising providinginstructions to visualize the region in a time period about two weekssubsequent the administration.
 41. The method of claim 35, comprisesproviding instructions to prepare at least one of the effective amountand the effective concentration based in part on a diameter of thetarget area of the biological vessel.
 42. The method of claim 35,wherein at least one of the effective amount and the effectiveconcentration is based in part on the flow rate of the blood in thebiological vessel.
 43. The method of claim 35, wherein at least one ofthe effective amount and the effective concentration is based in part onproviding a hydrogel having an average pore size that is less than anaverage diameter of a red blood cell of the subject.
 44. The method ofclaim 35, wherein the concentration effective to allow at least partialblockage of the biological vessel comprises a concentration in a rangeof about 0.1 weight percent to about 3 weight percent peptide.
 45. Themethod of claim 35, wherein the amount effective to allow at leastpartial blockage of the biological vessel comprises a volume in a rangeof about 0.1 mL to about 5 mL.
 46. The method of claim 35, furthercomprising providing instructions to monitor the area surrounding the atleast partial blockage to determine cell necrosis.
 47. The method ofclaim 35, wherein the formed blockage is used in the treatment ofdisorders, malformations, or congenital ailments in biological vessels.48. The method of claim 35, wherein the formed blockage is used in thereduction of cancerous cells.
 49. The method of claim 35, wherein thepeptide solution is substantially free of cells.
 50. The method of claim35, wherein the peptide solution is substantially free of drugs.
 51. Themethod of claim 35, wherein the subject is a mammal.
 52. The method ofclaim 51, wherein the subject is human.
 53. The method of claim 35,wherein the peptide has an amino acid sequence of one ofRADARADARADARADA (SEQ ID NO: 7), IEIKIEIKIEIKI (SEQ ID NO: 8), andIEIKIEIKIEIKIEIKI (SEQ ID NO: 9).